Urinary tract infections (UTIs) are most frequently caused by Escherichia coli and are common among women of all ages. Recent increases in antimicrobial resistance among uropathogens have stimulated interest in alternative, non-antimicrobial methods of preventing and treating UTI in lieu of antibiotics. UTI is preceded by specific alterations in the vaginal flora. Normally dominated by Lactobacillus species, the vaginal microniche becomes colonized with E. coli prior to the development of UTI. Depletion of vaginal lactobacilli is associated with vaginal colonization with E. coli and other pathogens, increasing the risk of UTI and certain sexually transmitted diseases. An ongoing clinical study is investigating the efficacy of a vaginal Lactobacillus suppository, L crispatus CTV-05, in preventing recurrent UTI (DK53369). However, the mechanisms that mediate protection by probiotic strains of Lactobacillus in the vagina have not been systematically investigated. The central hypothesis of this proposal is that vaginal lactobacilli such as this probiotic isolate L crispatus CTV-05 utilize specific mechanisms in protecting the vaginal epithelium against pathogens such as uropathogenic E. coli. We will investigate the following specific hypotheses, using L. crispatus CTV-05, additional isolates from our collections, and primary cultured vaginal epithelial cells: (1) Lactobacilli adhere to the vaginal epithelium using specific adherence molecules and protect against adherence of uropathogenic E. coli via competitive inhibition, including by binding to common globoseries GSL receptors on vaginal epithelial cells (VECs). Adherence may also be mediated via S-layers; (2) Lactobacilli kill uropathogenic E. coli in vitro via production of HZO2 and bacteriocin-like substances and inhibits growth of uropathogenic E. coli via co-aggregation; and (3) Lactobacilli induce repair and barrier function genes upon adherence to vaginal epithelium, in contrast with pathogens such as E. coli, which induce proinflammatory genes. These studies will have important implications for understanding mechanisms of protection by normal flora and probiotics in the vaginal microniche and at other epithelial surfaces such as the gastrointestinal and upper respiratory tracts and will suggest rational design and/or enhancement of probiotics.